The present invention relates to a diagnostic method and kits for neuropsychiatric diseases using trinucleotide repeats sequences. More particularly, the present invention relates to using gene amplification methodology and reverse dot-blot and oligonucleotide probe technology. The methods and probes of the invention specifically relate to the detection of the SCA III genes. The invention relates to the fields of molecular biology, diagnostic medicine.
Because of their unclear causes and nosagenic mechanisms, neuropsychiatric diseases are very difficult to diagnose and treat. In recent, some neuropsychiatric diseases have been found to be associated with characteristic genes. Accordingly, extensive research has been made to take advantage of the genes in diagnosing the diseases accurately.
Trinucleotide repeats (TNR) sequences are microsatellite DNA which occur as tandem repeats of a 3-bp sequence in the genome. These microsatellites are of special interest in human genetics, because they vary in length among individuals, making them highly useful for gene mapping as well as for clinical diagnosis or prognosis of some genetic diseases, especially neuropsychosis. For example, normal individuals have tens of copies of TNR in their genomic DNA. For patients suffering from neuropsychosis, the number of certain TNR was reported to be increased several to hundreds times as many as that of normal individuals.
In 1991, it was first reported that the number of the repeat (CGG)n are increased in the genome of fragile X syndrome patients. Since then, many reports have been published of such diseases associated with trinucleotide expansion, inclusive of Friedreich's ataxia in 1996, SCA VI syndrome and SCA VII syndrome in 1997. As many as 13 diseases are now found to be associated with trinucleotide repeat expansion.
A study of fragile X syndrome in which the chromosome Xq 27.3 (FRAXA) locus of the patients was cloned, revealed that the increased number of FRAXA is a common phenomenon to the patients (Yu, S. et al., Science, 252:1179-1181, 1991). In addition, such an expansion of a trinucleotide repeat is found in individuals affected with myotonic dystrophy, causing an instable mitosis which leads to somatic mosaicism.
Besides, examples of the diseases associated with the expansion of TNR include Huntington's disease, spinocerebellar ataxia type I (SCA I), X-linked spinobular atrophy (SBMA), dentatorubral and pallidoluysian atrophy (DRPLA), spinocerebellar ataxia type III (SCA III) and FRAXE mental retardation.
Recently molecular genetics have provided good evidence that some genetic neuropsychosis is primarily attributed to an increase in the number of TNR. The genes comprising TNR are thought to be expressed as kinases which play essential roles in the brain, but the correlation of TNR and nosagenic mechanism has not been clearly unveiled yet.
The diseases attributed to the TNR expansion show relatively common clinical options. In detail, there are four common clinical traits in such diseases: phenomenon of anticipation, inheritance disposition (autosomal dominant and sex chromosomal dominant), neural regression or mental retardation, and somatic mosaicism.
The phenomenon of anticipation is the pattern in which genetic diseases seem to display an earlier age of onset and more severe expression in more recent generations of a pedigree. That is, the degree of severity increases in each generation. For example, myotonic dystrophy is expressed differently in each generation of a three-generation family. Grandfather expresses the disease in the sixties or seventies, father in the thirties or forties, and grandson in the teens. DNA sequence analysis showed that a CGG repeat unit is present in 50 or less copies in normal individuals. Those with fragile X syndrome have 200 or more CGG repeats. An intermediate number of repeats, ranging approximately from 60 to 200, is seen in normal transmitting individuals, who carry the gene but do not express the disease, that is, who are of normal intelligence with no observation of the appearance characteristic of the patients. When the normal transmitting individuals transmit the gene to their offspring, there is often an expansion from 60-200 repeats to much more than 200 repeats. In addition to mental retardation, fragile X syndrome is characterized by an abnormal facial appearance with large ears, and macroorchidism in postpubertal males. This is observed in most of the diseases associated with TNR expansion, so that the prognosis of the diseases is possible.
As an example of the so-called dynamic mutation in genetics, autosomal dominant or sex chromosomal dominant inheritance is the phenomenon in which the genetic information which should remain as it is for continuous generations, is changed in just one next generation.
Neural regression or mental retardation is the very reason why the patients affected with the diseases go to hospital. As leading symtoms, metal retardation is shown in fragile X syndrome or FRAXA syndrome, muscular strength reduction resulting from progressive muscle deterioration in mytonic dystrophy, and ataxia in other diseases. Hence, the neuropsychiatric diseases can be diagnosed by investigating the genes of the patients who show the symptoms.
Somatic mosaicism, a characteristic trait of neuropsychosis, is detected by southern hybridization and methylation analysis with the gene of, for example, a fragile X syndrome patient or his family, whose result is a smeared band pattern in addition to a normal single band, showing that the increasing behavior of the number of the CGG repeat unit is different from generation to generation. This is attributed to the fact that the number of a CGG repeat unit is differently increased upon mitosis as well as meiosis. Also, a patient with fragile X syndrome was reported to have different numbers of the CGG repeat unit from organ to organ. This makes it difficult to accurately diagnose fragile X syndrome before birth through chorionic villous sampling. In fact, it was reported that methylation abnormality is not detected by the pre-parturient diagnosis while being evident in a peripheral blood test.
The neuropsychiatric diseases caused by an increase in the number of TNR have a common phenomenon that the sequence is repeated at the same gene locus of almost all patients. The gene mutation which is a cause for neuropsychiatric diseases seems to originate from the same ancestor (founder effect) because it is attributable to abnormality in a specific gene locus. In contrast, the mutation which causes Marfan syndrome or hereditary hypertrophic cardiomyopathy depending on autosomal dominent disease is relatively widely spread on many exons. Very exceptionally, there was found a fragile X syndrome which was caused by a mutation in an exon rather than by the TNRsequence. However, almost all neuropsychiatric diseases are caused by the mutation collected in one locus.
Linkage analysis demonstrated that the gene causing the neuropsychosis contains an increased number of TNR to give the same polymorphic marker on the chromosome.
By taking advantage of the fact that the neuropsychiatric diseases are caused by the simple mechanism of an increase in the number of TNR on a certain gene, they can be clinically diagnosed. That is, the diseases can be detected at high accuracy by investigating the increased degree of the number of TNR rather than all exonal mutations. Conventionally, fragile X syndrome was judged with an accuracy of 50% by investigating the X chromosomes from the cells of suspicious individuals, which are cultured in a medium deficient in folic acid.
Searching methods of the TNR associated the neuropsychosis were already developed and patented (U.S. Pat. Nos. 5,545,539, 5,650,277, 5,597,694, 5,723,301 and 5,582,979, and WO 97/27328). Particularly, the gene associated with Huntington's disease itself is patented (General Hospital Corporation; U.S. Pat. Nos. 5,538,844, 5,686,288 and 5,693,757 and EP No. 0 814 977). However, nowhere has been reported methods for diagnosing neuropsychiatric diseases through the reverse dot hybridization or polymerase chain reaction-microplate hybridization (PCR-MPH) using gene loci containing TNR.
Application of these molecular genetic methods for neuropsychiatric diseases will guarantee highly accurate diagnosis. Particularly, reverse dot hybridization, a modified dot hybridization method in which after a probe is immobilized on a membrane or a plate, target DNA is hybridized with the probe and the hybrid is quantitated, is anticipated to be useful as a routine screening assay in hospitals, by virtue of the employment of many probes on one plate or membrane and a simple procedure.
An HLA typing method using PCR and reverse dot hybridization was developed and patent was filed (Japanese Pat. Appl'n No. Heisei 8-83480) by Wakunaga company of Japan. However, use of such a method in the diagnosis of neuropsychosis has not been reported at all.